The present invention relates to a method for isolating at least one analyte from a liquid medium, in which the latter is distributed.
xe2x80x9cIsolatingxe2x80x9d or xe2x80x9cisolationxe2x80x9d is generically understood to mean any technique which makes it possible to separate said analyte, but also to enrich or concentrate, in relation to said analyte, any liquid mixture containing it. However, it is also understood to mean, optionally conjointly with the preceding definition, any technique which makes it possible to determine the analyte, for the purpose of a detection and/or quantification thereof, in the liquid medium containing it.
xe2x80x9cAnalytexe2x80x9d is understood to mean any entity, in particular biological entity, to be isolated. Among the range of the analytes considered below by the present invention, there may be mentioned cells, organelles, viruses and bacteria, antibodies, antibody fragments, antigens, haptens, lectins, sugars, ribodeoxyribo-nucleic acids, proteins, in particular A or G, hormones, hormone receptors, biotin, avidin, streptavidin and in general all natural or synthetic molecules or macromolecules, or analogs, to be determined, that is to say detected and/or quantified.
In accordance with the document FR-A-2,679,660, a method is known for isolating at least one analyte, namely either an antigen which is free or bound to the surface of a cell, or an antibody directed against a cellular or tissue antigen, for example anti-erythrocyte antibody.
According to this method, at least one reagent is available which comprises, on the one hand, a free support, in discrete form, in this case relatively large-sized magnetic particles consisting of a polymer which encapsulates ferrite granules, and, on the other hand, exhibiting a receptor for the analyte, namely either an antibody, or an antigen or revealing substance, for example an anti-human immunoglobulin antibody.
Another support, for capture, is also available which consists of the wall of a container or well, whose accessible surface comprises at least one useful or active zone, of limited surface, exhibiting at least one other receptor for an entity to be captured, namely for said analyte or for said receptor. This other receptor is either an antibody, or an antigen, for example of the erythrocyte type.
According to this method, the accessible surface of said other support is brought into contact with the analyte, contained in a liquid sample, in order to bind the analyte to the accessible surface, and to assemble it in the form of a deposit on said surface, immobilized on said other support via said other receptor.
Next, for revealing purposes, the reagent is brought into contact with the immobilized analyte, in order to obtain with the latter a combination, also immobilized on said other support and capable of allowing the isolation or determination of the analyte.
A method as defined above is therefore carried out with a reagent consisting of a support in discrete form, to which at least one ligand forming a receptor for the analyte is bound.
Preferably, these particles are magnetic. They can be classified into two categories, namely particles of relatively large diameter, for example of the order of one or a few micron(s), and those of relatively small diameter, for example of the order of a few tens of nanometers, and in the colloidal state.
The magnetic particles of relatively large diameter, when they are placed in a magnetic field, move in the direction of the site where the field is highest and at a sufficient speed to be separated from their medium by this means.
By way of example, there may be mentioned the particles described in the document EP-A-0,125,995. They are obtained by precipitation of ferrous and ferric salts in basic medium, followed by a silanization reaction in methanol. Their final diameter is between 0.1 and 1.5 xcexcm and their density is 2.5 g/cm3. Likewise, the particles described in the documents EP-A-0,106,873, EP-A-0,585,868 and U.S. Pat. No. 5,356,713 are obtained by various methods of polymerization, or alternatively those described in the document U.S. Pat. No. 4,297,337 use a porous glass matrix in which magnetic pigments are dispersed. Other patents also describe the use of small-sized particles, but deliberately aggregated in order to increase the magnetic mass as in the document U.S. Pat. No. 5,169,754. The article by P. A. Risxcfx86en et al, Protein Expression and Purification, 6 (1995), 272-277 also describes magnetic gels.
Placed in a magnetic field, all these relatively large particles generate a movement in the direction of the side where the field is the most intense. A simple permanent magnet or equivalent assemblies as described for example in the document EP-A-0,317,286 or U.S. Pat. No. 565,665 may be used. These particles are commonly used for the separation of cells or of molecules, as well as in immunoassays as described in the document EP-A-0,528,708 but are not used as systems for detection and quantification.
Moreover, their diameter and their density are such that they sediment very rapidly under the effect of gravity, which makes them difficult to use because of the stirring stresses or lack of homogeneity of the solutions due to the creation of a concentration gradient.
In summary, the relatively large particles are difficult to use and are not very appropriate for the determination of an analyte.
Magnetic particles of relatively small diameter are practically not attracted by a simple permanent magnet within reasonable periods: these particles are in particular widely used for the magnetic separation of cells. These particles are known to persons skilled in the art by the name of xe2x80x9csuperparamagneticxe2x80x9d particles. xe2x80x9cSuperparamagneticxe2x80x9d particles are understood to mean particles whose diameter is too small to consist of several magnetic domains. They are characterized by a high magnetic susceptibility and a high magnetization at saturation, but a zero or very low magnetic stability. These particles are in particular widely used for the magnetic separation of cells. For example, those described in the document U.S. Pat. No. 4,230,685 are obtained by emulsifying a mixture of albumin, of protein A and of particles of Fe3O4 15-20 nm in diameter and can immobilize antibodies via the protein A. The document U.S. Pat. No. 4,452,773 describes another type of particle, obtained by precipitation of ferrous and ferric salts in a basic medium, and in the presence of a polysaccharide. These particles can immobilize antibodies, oligonucleotides, lectins or other biomolecules, by coupling to the polysaccharide by means of known methods of grafting. Their use has often been repeated, as in the documents U.S. Pat. No. 5,543,289 or WO-A-88/00060, or they are used in specific applications such as those described in the documents FR-A-2,710,410 and FR-A-2,732,116. The document U.S. Pat. No. 4,795,698 describes a modification of the Molday procedure, by replacing, for example, the polysaccharide with another polymer of a protein nature. The proteins present at the surface of the particles can thus serve for the subsequent immobilization of antibodies by coupling methods known to persons skilled in the art.
These particles require the use of special assemblies which make it possible to locally increase the magnetic field gradient. This technique is known by persons skilled in the art by the name of HGMS (for High Gradient Magnetic Separation) and it is for example described by WO 96/09409. It uses a device discrete form and for this reason are not used for the separation, concentration or enrichment of an analyte.
Furthermore, after separation, the particles are rather considered as an inconvenience in the subsequent steps of any method. In the document U.S. Pat. No. 4,018,886, they are even removed deliberately. The document FR-A-2,710,410 uses the presence of small-sized superparamagnetic particles as detection components which make it possible to quantify a molecular recognition reaction. In this case, the principle used is an agglutination reaction resulting in the formation of an aggregate. Furthermore, the articles are not used as a means of concentrating or of separating the component to be separated, but solely as a means of detection.
In summary, relatively small magnetic particles are not very appropriate for separation, enrichment or concentration procedures, in traditional procedures, in particular immunoassays.
The document WO-A-96/09409 describes the use of the so-called MACS (Magnetic Cell Sorting) method, involving the formation of a complex between an analyte and a reagent comprising magnetic particles, for enriching/concentrating said analyte, in this instance fetal erythrocytes. These cells are then analyzed by flow cytometry, or used as a source of genetic material, but without being immobilized on a support, through bonding between the analyte and a receptor for example.
The document DE-A-4,036,288 describes a method which makes it possible to detect and quantify one or more analytes specifically bound to particles which are capable of being distinguished.
The subject of the present invention is a method, which makes it possible to isolate an analyte from a liquid medium in which it is extremely dilute or not concentrated to any great extent. More particularly, as regards the determination of the analyte, the subject of the present invention is a method allowing a high sensitivity of detection and/or of quantification.
In accordance with the present invention, in general:
the reagent is brought into contact with a liquid sample obtained from the medium containing the analyte, to form an intermediate medium comprising, still in a discrete form and distributed in said medium, a complex between said support, the receptor and the analyte,
the accessible surface of said other support is brought into contact with at least one capture partner,
The deposit of the capture partner is concentrated by obtaining, from the intermediate medium, another sample, enriched with capture partner; and said other sample is brought into contact with the accessible surface.
A liquid stream, containing the capture partner, of limited section and adapted to the accessible surface, is established and said stream is brought into contact with said accessible surface.
A liquid stream, containing the capture partner, is established and said stream is brought into contact with said accessible surface, and then recycled in contact with the latter.
Before the description of the invention, it is appropriate to give a definition of the terms used.
xe2x80x9cLigandxe2x80x9d is understood to mean a component capable of forming, through a chemical or physical bond, a complex with an analyte.
By way of example of ligand, there may be mentioned antibodies, antibody fragments, antigens, haptens, lectins, sugars, ribo- and deoxyribonucleic acids, proteins, in particular A or G, hormones, hormone receptors, biotin, avidin or strepta-,idin and, in general, it being possible for natural or synthetic ligands, and modified ligand analogues to enter into competition with the ligands.
xe2x80x9cReceptorxe2x80x9d is understood to mean any ligand as defined above, immobilized on a support by any means such as adsorption, covalent bonding, chelation, molecular recognition, and the like, and capable of retaining an analyte, alone or conjugated with another ligand.
xe2x80x9cSupportxe2x80x9d is understood to mean any type of support, polymeric, inorganic or metallic. By way of example of polymeric supports, there may be mentioned plastic supports based on polystyrene, poly(meth)-acrylates, polybutadiene, polypropylene, and the like, alone or in the form of copolymers. By way of example of inorganic supports, there may be mentioned silicon oxide, silicon, mica, glass, quartz, and the like. By way of example of metallic supports, there may be mentioned gold, silver, titanium oxide, vanadium oxide, and the like.
The immobilization of the ligands on the support may be carried out either by simple adsorption on the native or modified support, or via a chemical or physical reaction which makes it possible to modify the surface of the support, and thus to allow the binding of the receptor by covalent bonds, or other traditional means well known to persons skilled in the art.
xe2x80x9cLimited surfacexe2x80x9d is understood to mean any surface obtained by chemical or physical means, designed to reduce a surface of defined dimensions, for example by cutting into smaller-sized components, by covering with a xe2x80x9cmaskxe2x80x9d which limits the initial surface to the inner contours of the mask, or by chemical means of spreading based on a smaller surface, as described in Kumar A. et al. (Langmuir (1994), 10, 1498-1511). The reduction in the surface of the useful or active zone is not limited in size. Methods derived from microtechnologies make it possible, for example, to obtain limited surfaces of micro or nanoscopic sizes, as well as to have available and/or to convey micro or nanovolumes of liquids to said useful zone of limited surface.
In the description below, the term xe2x80x9cconjugatedxe2x80x9d will be reserved for the entity formed by immobilization between the ligand and the support of the reagent. The term xe2x80x9ccomplexxe2x80x9d will be reserved for the entity formed between the reagent, hypothetically conjugated, and the analyte. Whether it is the support which is part of the reagent, the reagent itself, and the complex, they are in a discrete form, distributed or dispersed in a liquid medium, and consequently in the form of particles.
The terms xe2x80x9csupportxe2x80x9d and xe2x80x9cother supportxe2x80x9d indicate that they are components which are substantially of the same nature or having essentially the same function. The term xe2x80x9csupportxe2x80x9d will be reserved for the support belonging to the reagent, and the term xe2x80x9cother supportxe2x80x9d, to the support of which at least part of the accessible surface or window contains one or more useful zones of limited surface.
The terms xe2x80x9creceptorxe2x80x9d and xe2x80x9cother receptorxe2x80x9d will be reserved respectively for the ligands immobilized respectively on said support and said other support.
xe2x80x9cParticlexe2x80x9d is understood to mean any particle of a polymeric, inorganic or metallic support, on which it is possible to graft a ligand. In particular, the particles which may be separated by the action of an external physical means, for example by a magnetic or electrical route, or under the effect of gravity or by centrifugation are considered as falling within the scope of the present invention. Falling outside the preceding definition are the small-sized, in particular superparamagnetic, particles whose speed of sedimentation under the effect of gravity is less than the thermal agitation, but which are capable of constituting aggregates by any method which makes it possible to combine them with each other, or to assemble them on larger-sized particles, which can be separated by any physical means.
By way of example of polymeric particles, there may be mentioned the particles obtained by polymerization in emulsion such as latexes or larger-sized particles, magnetic or otherwise.
By way of example of metallic particles, there may be mentioned colloidal gold, ferro-, ferri-, para-or superparamagnetic particles, coated or otherwise with natural or synthetic polymers, whose composition comprises iron or other metals such as cobalt, nickel, alone or in the form of alloys, magnetic or otherwise.
By way of example of inorganic particles, there may be mentioned particles based on silica or silicon, magnetic or otherwise.
By way of example of methods of separation by an external physical means, there may be mentioned sedimentation by gravity or by centrifugation, magnetic attraction by the action of permanent magnets, of electromagnets, or by the use of devices which make it possible to increase the magnetic field gradient, the electrical attraction, and any other equivalent technique.
xe2x80x9cDeterminationxe2x80x9d is understood to mean any method which makes it possible to detect the presence and/or to quantify the deposit concentrated in the useful zone of the accessible surface of said other support, namely the particles of the specific product of interest, that is to say of the complex or the reagent which has not reacted.
By way of example of a method of determination, there may be mentioned:
topographical methods such as atomic force microscopy, profilometry, and the like,
magnetic methods as described, for example, in the document FR-A-2,710,410, magnetic force microscopy, and any methods of determination which are sensitive to the presence of ferro, ferri, para and super paramagnetic metallic components
electrical methods such as the measurement of a variation in capacitance, described for example in the document EP-A-90 402 611, tunnelling microscopy, impedance measurements, and the like
optical methods which allow the detection, for example, of a modification of the thickness and/or of the refractive index (optical thin layers, ellipsometry, surface plasmon resonance, surface acoustic waves, and the like) or the measurement of a light intensity (evanescent waves, dark field microscopy, optical near field microscopy, and the like)
methods which allow the measurement of mass variations (quartz crystal microbalance, and the like)
and in general, any techniques not cited here, but which are equivalent.
The present invention can accomodate various embodiments, depending on the operating choices defined below.
In the concentration step, the capture partner concentration level is determined so that the quantity of said immobilized capture partner remains at most equal to the quantity of said other receptor on said other support. To this end, for example, the reagent comprises particles of the support conjugated with the receptor, having the capacity to be separated from any liquid medium in which they are dispersed, under the action of a physical means applied to the liquid medium, and this physical means is applied to at least part of the intermediate medium, in order to separate the complex particles, these complex particles thus separated are optionally washed, and the complex particles are recovered, by ending the application of the physical means, in order to subsequently obtain, in said useful zone, a deposit concentrated in relation to the capture partner. By way of example, the particles of the support are magnetic, in particular superparamagnetic, and the physical means consists of a magnetic field applied to the intermediate medium, for example according to the so-called HGMS (High Gradient Magnetic Separation) technique.
The receptor, that is to say the one belonging to the reagent, and said other receptor, that is to say the one for the accessible capture surface, contain the same ligand, immobilized respectively on the support and said other support.
When the reagent comprises particles of the support conjugated with the receptor, having the capacity to be separated from any liquid medium in which they are dispersed, under the action of a physical means applied to this liquid medium, this physical means is preferably applied in relationship with the other support, that is to say the one for capture, in order to concentrate said deposit in any useful zone.
In some cases, the reagent is labeled, and the treatment of the analyte comprises a step of determining the marker for the deposit concentrated, immobilized in the useful zone of the other support, that is to say of the capture support. Conventionally, this labeling of the reagent is obtained according to one of the following modes, namely:
the support itself constitutes a marker; it is for example colloidal gold
the marker is bound to the support
the marker is bound to the receptor.
The analyte is determined from the deposit concentrated in the useful zone, by any method chosen from the group including topographical methods, for example by atomic force microscopy (AFM), magnetic methods, for example by magnetic force microscopy, electrical methods, for example measurement of a variation in capacitance, optical methods, for example measurement of the refractive index, and finally methods for measuring mass variation, for example quartz crystal microbalance.
More particularly, the useful zone is designed so that the quantity of said deposit immobilized on said other support is at least equal to the sensitivity threshold of the method of determination, expressed as number of particles per unit of surface.
The assay format used is either direct, in which case said capture partner is said analyte, or by competition, in which case said capture partner is said reagent.
Preferably, another sample is obtained, by enriching said intermediate medium with complex, and said other sample is brought into contact with the accessible surface of said other support.
Various modes of bringing the other sample into contact with the accessible capture surface are considered:
a measured quantity of said other sample is deposited on the accessible surface of the latter
or a stream of said other sample, of a relatively small thickness, optionally recycled, is caused to pass in contact with this accessible surface.
Finally, the liquid medium may contain a plurality of various analytes, and in this case the accessible capture surface of the other support comprises a plurality of useful zones, exhibiting a plurality of other receptors which are respectively different, of capture partners which are respectively different.
The method according to the invention may be applied to a specific nucleic material of any bacterium, in this instance ribosomal RNA, in which case the sensitivity level obtained makes it possible to avoid any enzymatic amplification method, such as PCR (Polymerase Chain Reaction), applied to the genomic material of the same bacterium, in order to detect the latter. Indeed, the sensitivity level obtained becomes compatible with the quantity of ribosomal RNA present in the bacterium, of the order of 104 to 105 molecules, which makes it possible to determine the presence of this bacterium, without using amplification of all or part of its genome.